Differentiation of "Trypanosoma cruzi", "T. cruzi marinkellei", "T. dionisii" and "T. vespertilionis" by monoclonal antibodies

Volltext

(1)Differentiation of "Trypanosoma cruzi", "T. cruzi marinkellei", "T. dionisii" and "T. vespertilionis" by monoclonal antibodies. Autor(en):. Petry, K. / Baltz, T. / Schottelius, J.. Objekttyp:. Article. Zeitschrift:. Acta Tropica. Band (Jahr): 43 (1986) Heft 1. PDF erstellt am:. 28.01.2022. Persistenter Link: http://doi.org/10.5169/seals-313606. Nutzungsbedingungen Die ETH-Bibliothek ist Anbieterin der digitalisierten Zeitschriften. Sie besitzt keine Urheberrechte an den Inhalten der Zeitschriften. Die Rechte liegen in der Regel bei den Herausgebern. Die auf der Plattform e-periodica veröffentlichten Dokumente stehen für nicht-kommerzielle Zwecke in Lehre und Forschung sowie für die private Nutzung frei zur Verfügung. Einzelne Dateien oder Ausdrucke aus diesem Angebot können zusammen mit diesen Nutzungsbedingungen und den korrekten Herkunftsbezeichnungen weitergegeben werden. Das Veröffentlichen von Bildern in Print- und Online-Publikationen ist nur mit vorheriger Genehmigung der Rechteinhaber erlaubt. Die systematische Speicherung von Teilen des elektronischen Angebots auf anderen Servern bedarf ebenfalls des schriftlichen Einverständnisses der Rechteinhaber. Haftungsausschluss Alle Angaben erfolgen ohne Gewähr für Vollständigkeit oder Richtigkeit. Es wird keine Haftung übernommen für Schäden durch die Verwendung von Informationen aus diesem Online-Angebot oder durch das Fehlen von Informationen. Dies gilt auch für Inhalte Dritter, die über dieses Angebot zugänglich sind.. Ein Dienst der ETH-Bibliothek ETH Zürich, Rämistrasse 101, 8092 Zürich, Schweiz, www.library.ethz.ch http://www.e-periodica.ch.

(2) Acta Tropica 43, 5-13 (1986). Department of Protozoology. Bernhard-Nocht-Institute for Nautical and Tropical Medicine. 2000 Hamburg. Federal Republic of Germany laboratoire d'Immunologie et de Biologie parasitaire. Université dc Bordeaux II. 33076 Bordeaux. '. France-. Differentiation of Trypanosoma cruzi, T. cruzi marinkellei, T. dionisii and T. vespertilionis by monoclonal antibodies K. Petry1, Th. Baltz2,. J.. Schottelius1. Summary. Anti-T. dionisii and anti-T. vespertilionis monoclonal antibodies secreted hybridoma clones were tested against various strains of T. dionisii. T. vespertilionis, T. cruzi and T. cruzi marinkellei. Strain and species specific antigens were detected for the homologous immunizing strains. The common antigenic determinants of the tested trypanosome species include a component of the flagellum and different cell structures. Seventeen T. cruzi strains could be classified into two groups when tested with anti-J1, dionisii monoclonal antibodies. The cross reactions between T. dionisii and T. cruzi demonstrate a strong correlation between T. dionisii and T. cruzi group 2. On the other hand T. cruzi group and T. cruzi marinkellei show very similar antigenic character. by 17. 1. Key words: Trypanosoma cruzi; T. cruzi marinkellei; T. dionisii; T. vespertilionis;. monoclonal antibodies.. Introduction Bat trypanosomes of the subgenus Schizotrypanum have a cosmopolitan distribution. Biometrie studies and morphological investigations (Hoare. 1972; Mühlpfordt, 1981) have shown that they are comparable to the etiologic agent of Chagas' disease, Trypanosoma cruzi, which infects 20 million people in endemic areas in Latin-America (WHO, 1983). Bat trypanosomes ofthe Old World are host restricted to bats only, but some American stocks of bat trypanosomes are Correspondence: Klaus Petry. BNI D-2000 Hamburg 4. FRG. -. Department of Protozoology. Bernhard-Nocht-Strasse 74..

(3) infective to laboratory animals and presumably transmissible by Triatominae bugs (Hoare, 1972; Marinkelle, 1982). Using host specificity as a differentiating character is problematic because T. cruzi strains of low virulence have been found (Hüther, 1981). Therefore, an accurate identification of Schizotrypanum species of bats is important for epidemiological studies of T. cruzi. Several techniques have been used to differentiate bat trypanosomes: buoyant density of DNA, starch gel electrophoresis (Baker et al.. 1978). isoelectrofocusing (Ebert, 1983) and lectins (Schottelius et al., 1983). Monoclonal antibodies have allowed distinction of T. cruzi from T. rangeli (Anthony et al, 1981 or differentiation of T. cruzi strains and clones (Kirchhoff et al, 1984a), and are useful for the development of precise diagnostic assays of T. cruzi strains and T. cruzi zymodemes (Flint et al, 1984). Furthermore, monoclonal antibodies have been used to characterize stage specific antigens of T. cruzi (Snary et al., 1981; Sher and Snary, 1982; Araujo et al. 1982) and to study their physiology (Alves et al., 1983). This work reports the differentiation ofthe bat trypanosomes T. dionisii and T. vespertilionis, and the agent of Chagas' disease. T. cruzi, by monoclonal antibodies raised against T. dionisii and T. vespertilionis. Materials and Methods Trypanosomes. The trypanosome strains of T. dionisii. T. vespertilionis. T. cruzi and T. cruzi marinkellei which were investigated are listed in Table 1. Epimastigote forms of these parasites were cultivated at 26 C in Brain-Heart-Infusion-Agar (Difco) containing rabbit blood.. Hybridoma techniques. Hyperimmunization was done with inbred Balb/c adult female mice inoculated intraperitoneally with live culture forms (5xl07 epimastigotes washed 3x with phosphate buffered saline. pH 7.4 [PBS]) of I. dionisii strain P3 and /. vespertilionis strain P14. The inoculation procedure was repeated 5xat an interval of 3 weeks. Five weeks after the last injection the mice were inoculated with 108 epimastigotes. Four days later, spleen cells from the boosted mice were fused with the X63 myeloma line following the strategy described by Fazekas de Sainl-Groth and Scheidcgger (1980). Screening. of monoclonal antibodies. Antibody production by hybrid cells was assessed between 10 and 14 days after fusion. Epimastigote trypanosomes were washed twice with PBS and fixed in 1% formaldehyde in PBS for two hours. Thereafter, they were washed 3x with PBS and used to prepare slides for indirect immunofluorescence (HF). Slides were air dried and kept at -20'C until used. Hybridoma supernatants were screened by I1F. The conjugate rabbit anti-mouse immunoglobulin labeled with fluoresceine isothiocyanate (FITC) (Institut Pasteur Production) was diluted in PBS (1:100) containing Evans Blue counterstain (1:10.000). All the anti-7". dionisii (dion) and anti-T. vespertilionis (vesp) monoclonal antibodies were tested by IIF several times with 3 T dionisii. 2 T vespertilionis. 17 T. cruzi and 2 T. cruzi marinkellei strains..

(4) Table. 1.. Parasite strains used for the production* and characterization of monoclonal antibodies. Species/strain T.. Source. Locality. Host. England. East Anglia England. East Anglia England. East Anglia. Pipistrelli! pipi snellii P. pipislrelliis P. pipislrelliis. England. East Anglia England Rollcsby. Norfolk. P.. Brazil. Sào Paulo State Brazil. Sào Paulo State Brazil. Fazenda Velha Brazil. Bahia State Brazil. Bahia State Brazil. Rio Grande do Sul Mexico. Tehuantepec area Venezuela. San Juan Venezuela. El Yagual. Santa Rosa. Carabobo State Venezuela. La Pavona. San Francisco dc Asis, Aragua Stale Venezuela. Mollcjon. Portuguesa Slate Venezuela. Mollcjon, Portuguesa State Venezuela. Palambra del Doctor. Cojedes State Venezuela. Macuayas. Cojedes State Venezuela. La Coromoto. Cojedes State Venezuela. El Hoyon. Marinas State Venezuela. Oritz. Guarico State. patient 'Lodarla laticatidata patient patient Dulelphis azarac Triatoma infestans Triatoma sp. patient Diclclphis marsupial is. (2) (3) (4) (5) (4) (2) (6) (7) (7). Didelphis marsupialis. (7). Rhodnius prolixus. (7). Canis familiaris. (7). Rattus rutins. (7). patient. (7). Panstrongylus geniculatus. (7). Rhodnius robustus. (7). Triatoma maculata. (7). Brazil. Bahia State Brazil. Bahia State. Phyllostomum discolor P. discolor. (8) (8). dionisii. P2. P3* P7. s. s. (1) (1) (1). T vespertilionis P9. P14* 1.. 12-SF-2S. WA 301/130 MR Tehuantepec V OPS 4 OPS 6. OPS 9 OPS 12 OPS. 13. OPS. 21. OPS 22 OPS 53. OPS 89. B7 B9. pipislrelliis pipistrellus. (1) (1). cruzi. Y Morcego 1354 Esmeraldo. T.. P.. cruzi marinkellei. Dr. J. R. Baker. Medical Research Council Biochemical Parasitology Unit. Molteno Institute. Cambridge. U.K. 2. Prof. Dr. Z. Brener. Centro des Pesquisas René Rachou. Belo Horizonte. Brazil 3. Prof. Dr. M. P. Baretto. Fac. Pharm. Odont., USP, Ribeirào Preto. Sào Paulo State. Brazil 4. Dr. D. S. Ketteridge. London School of Tropical Medicine and Hygiene. London. U.K. 5. Prof. Dr. Z. Andrade. Fac. Med. UFBa. Salvador. Bahia State. Brazil 6. Department of Protozoology. BNI. Hamburg. F.R.G. 7. Research and Reference Center for Vector Biology and Control. Las Delicias. Maracay. Venezuela 8. Dr. M. A. Miles. London School of Tropical Medicine and Hygiene. London. U.K. 1..

(5) Results. In order to differentiate the epimastigotes of T. dionisii, T. vespertilionis and T. cruzi. monoclonal antibodies were raised against T. dionisii strain P3 and T. vespertilionis strain P14. The IIF method was chosen to detect the epimastigote antigens. Seventeen positive hybridomas were obtained: six directed against T. dionisii and eleven against T. vespertilionis. Cross reactions using different T. dionisii, T. vespertilionis, T. cruzi and T. cruzi marinkellei strains were carried out. Table 2 summarizes the results obtained with all tested trypanosome strains and indicates the existence of species specific and strain specific antigens. Nine (eight vesp and one dion) monoclonal antibodies were specific for the homologous immunizing strains. A species specific epimastigote antigen for all T. dionisii strains was revealed by dion 2.1a. This result demonstrates the homogeneity of the T. dionisii strains, while the two T. vespertilionis strains showed antigenic polymorphism, when tested with vesp 3.1 and vesp 4.3 monoclonal. antibodies. Three out of 17 monoclonal antibodies (vesp 9.1. vesp 9.3 and dion 10.1b) reacted with all trypanosome strains. A common antigen expressed only by T. vespertilionis and T. dionisii strains could be demonstrated by vesp 11.4. The 17 T. cruzi strains considered in this study could be classified into two groups when tested against two anti-T. dionisii monoclonal antibodies (dion l.ld and dion 4.6). 12 T. cruzi strains (group 1) did not react with either monoclonal antibodies, while the other 5 strains (group 2) reacted with both monoclonal antibodies. T. cruzi marinkellei strains B7 and B9 behaved very similarly to T. cruzi group 1, but they differed partly in cross reaction with monoclonal antibody dion 5.1b, which reacted with all T. cruzi strains. In fact. T. cruzi marinkellei strain B9 seems to be a variation ofthe T. cruzi group 1. The raised monoclonal antibodies reacted either with the cytoplasm or with the cell membrane or uniformly stained the whole parasite. Table 3 shows the results of these observations. As exemplified by strains Y (T. cruzi group 2), Tehuantepec (T. cruzi group 1) and B7 (T. cruzi marinkellei the fluorescence pattern of those monoclonal antibodies which caused cross reactivity was structurally similar to that obtained using the immunizing strains T. dionisii P3 and T. vespertilionis P14, although the intensity of fluorescence among the various tested parasite strains was different. Almost all monoclonal antibodies reacted with antigenic structures ofthe cell membrane. Two monoclonal antibodies (dion 10.1b and vesp 9.3) were specific for a common antigenic determinant present on the surface and in the cytoplasm of all T. dionisii. T. vespertilionis, T. cruzi and T. cruzi marinkellei strains. The monoclonal antibody vesp 9.1 recognized exclusively a common flagellar antigen expressed by all trypanosome strains. Four monoclonal antibodies stained the cytoplasm: vesp 3.1. vesp 7.1 and vesp 8.2 produced a.

(6) Ufi siuoiin.i.ids.is. fil l"6. l'6. i. pue. ë'8. iisiuoi/i. /. '9. isuieSe. 9'S posiE.i. CT. soipoqtiuE. VI dso. Vi. v. |Euopouoiu. qi'oi + +. jo viiotjiaads. SOipOqilUE. +. +. +. C9 + + + + +. qi's + + + + +. oqijo. 9T ]BU0|30U0t\. siss;. nx. uoip pit aouaasajonjjounuiuit. (uiejis. ¦. (uiEJts. lopaijuumii. (t Sutziunuiiui). loaaipu]. (Z. Suiziuniuiui) StUOI])l.l3dS3,t. dnojä). dnojS). aadaiuEnip^ oei/ioe. "i SUIEJ1S oiqsx. Op[E.I3lUS3. nsnioip. iznjj. j. ¦. Zd £j Id. pçii. -±. 6d. ^[j. h 9 6. le ce eç 68. SdO SdO SdO SdO SdO SdO SdO SdO SdO. VM. /. Il. CI. A. izn.u. j. A. cS-JS oSaajopv. zi. sn. izn.ij. \i ia 6e.

(7) Table 3. Types of IIF staining reaction observed wilh anti-'/'. dionisii and ami-/ monoclonal antibodies. Monoclonal antibodies. vespertilionis. Species. '/' dionisii. T. vc sper-. 1.. 7.. cruzi. tilionis. cruz /' (group D. cruzi (group 2). marinkellei. P3. P14. Tehuantepec. Y. B7. m. -. -. m. -. -. -. m. -. m. m. m. m. m. m m.. 1.. Strain. dion 1.1 d 2.1a 4.6 5.1b 6.3 10.1b. pe m. m m m.. c. vesp 2.4 3.1. 4.3 5.6 6.2. -. m. membrane; c. m. -. m m m c. -. -. fl. Il. m. c. m. c m m. m.. m. ccllbody: fl. flagellum: pe. m.. -. c. -. c. -. fl. 8.2 9.3 11.4 14.2. m.. c. 7.1. 9.1. m. c. -. c. -. -. -. Il c. m.. -. -. c. n c. m. c. -. cytoplasmic structure ofthe posterior end. homogenous fluorescence, while the T. dionisii specific monoclonal antibody dion 2.1a reacted exclusively with cytoplasmic microgranules at the posterior end of all T. dionisii strains.. Discussion. The difficulty of distinguishing trypanosome species and subspecies ofthe subgenus Schizotrypanum is a major restraint to progress in epidemiological studies on Chagas' disease. The absence of clear biometrie and morphological characteristics of Schizotrypanum species (Hoare, 1972; Marinkelle, 1976) does not allow easy differentiation between T. dionisii. T. vespertilionis and T. cruzi. Although the ultrastructure of kDNA can be used as a marker to differentiate T cruzi from T. rangeli, T. conorhini and T. lewisi (Mühlpfordt, 1975), this 10.

(8) method does not differentiate between different bat trypanosomes of the subgenus Schizotrypanum and T. cruzi (Mühlpfordt. 1981). T. dionisii could be distinguished from other bat Schizotrypanum species by the presence of long thin trypomastigotes in culture (Baker et al.. 1978). However, this form may also be found in T. vespertilionis cultures after agglutination with an epimastigote stage specific monoclonal antibody (data not shown). Isoenzyme patterns permit the differentiation between T. cruzi and T. vespertilionis. T. dionisii shows isoenzyme profiles identical with those of /'. cruzi group 2. T. cruzi group 1 is partly identical with T. cruzi marinkellei (Ebert. 1983).. Strains of T. cruzi and T. vespertilionis can be distinguished on the basis of analysis of surface carbohydrate determinants (Schottelius et al., 1983). Monoclonal antibodies against T. cruzi and other Schizotrypanum species are ideal tools to study the epidemiology of Chagas' disease. T. cruzi zymodeme specific antigens (Flint et al., 1984) and T. cruzi and T. rangeli specific antigens (Anthony et al. 1981). could be defined with the aid of monoclonal antibodies. The large degree of polymorphism among trypomastigote surface antigens of T. cruzi (Plata et al.. 1984) is a further indication ofthe highly heterogenous nature of T. cruzi strains which has been reported earlier in epimastigote culture forms using different methods: isoenzyme profiles (Miles et al.. 1977: Ebert. 1982a. b). lectins (Schottelius and Uhlenbruck. 1983) and monoclonal antibodies (Kirchhoffet al.. 1984a: Flint et al., 1984). Differences between T. cruzi strains and clones using a monoclonal antibody recognizing a carbohydrate epitope on a 72.000 m. w. glycoprotein expressed on the surface are probably based on structural changes or differences in the membrane environment ofthe molecule (Kirchhoff et al.. 1984b). On the basis of cross reactive anti-T. dionisii monoclonal antibodies 17 T. cruzi strains could be divided into two groups. These results are in agreement with previous isoenzyme analysis (Miles et al. 1981: Ebert, 1982a. b) and lectin typing (Schottelius. 1982; Schottelius and Uhlenbruck, 1983; Schottelius et al., 1983). T. cruzi group correlated with the zymodeme ZI or isoenzyme group and the PNA-type as investigated for Venezuelan T. cruzi strains (Mühlpfordt 1. 1. et. al. 1984).. Five out of six T. dionisii monoclonal antibodies cross reacted with T. cruzi group 2. These results confirmed the close relationship between T. cruzi group 2 and T. dionisii as was demonstrated by isoelectrofocusing (Ebert. 1983). T. cruzi marinkellei was correlated to T. cruzi group (Ebert. 1983). We could demonstrate by studying epimastigote antigens that strain B9 of this T. cruzi subspecies isolated from a bat was different from T. cruzi. as has been detected by DNA bouyant density, non-infectivity to mice and low infectivity to Triatominae bugs (Baker et al.. 1978). However. T. cruzi marinkellei strain B7 appeared to be similar to T. cruzi group 1. The present comparative study demonstrated that species specific mono1. 11.

(9) clonal antibodies are useful to distinguish T. cruzi from other trypanosomes of the subgenus Schizotrypanum. Because of their close relationship it will be necessary to test anti-7". cruzi monoclonal antibodies in epidemiological studies against T. dionisii. Acknowledgments. This paper shows partial results of a dissertation directed by Prof. Dr. H. Mühlpfordt. This prepublication is authorized by Biology PhD-Council at the University of Hamburg. F.R.G. K. Petry was supported bv a fellowship ofthe French Government/University of Bordeaux II and DAAD (German Academic Exchange Service). Bernhard-Nocht-Institute. Hamburg.. Alves M.J. M.. Kawa M. A.. Nussenzweig R. S.: Monoclonal antibodies of Trypanosoma inizi inhibit motility and nucleic acid synthesis of culture forms. Infect. Immun. 39. 377-382 (1983). Anthony R. L.. Cody T. S., Constantine N. T.: Antigenic differentiation of Trypanosoma cruzi and 'Trypanosoma rangeli by means of monoclonal hybridoma antibodies. Amer. J. trop. Med. Hyg. 30. 1192-1197 (1981). Araujo F. G.. Sharma S. D.. Tasi V.. Cox P.. Remington J. S.: Monoclonal antibodies to stages of Trypanosoma cruzi: characterization and use for antigen detection. Infect. Immun. 37. 344-349 (1982). Baker J. R.. Miles M. A.. Godfrey D. G, Barrett T. V.: Biochemical characterization of some species of Trypanosoma (Schizotrypanum) from bats (Microchiroptera). Amer. J. trop. Med. Hyg. 27. 483-491 (1978). Ebert F.: The identification of two main-groups of Trypanosoma cruzi stocks from Brazil by their isoenzyme patterns of isoelectrofocusing. Tropenmed. Parasil. 33. 140-146 (1982a). Ebert F.: The use of isoelectrofocusing in thin layer Polyacrylamide and agarose gels as a method for the characterization of Venezuelan Trypanosoma cruzi stocks. Tropenmed. Parasit. 33. 63-67 (1982b). Ebert F.: Comparison of isoenzymes of some species ofthe subgenus Schizotrypanum from bats by isoelectrofocusing. Tropenmed. Parasit. 34, 93-97 (1983). Fazckas de Saint-Groth S., Scheidcgger D.: Production of monoclonal antibodies: strategy and tactics. J. Immunol. Meth. 35. 1-21 (1980). Flint J. E.. Schechter M.. Chapman M. D.. Miles M. A.: Zymodeme and species specificities of monoclonal antibodies raised against Trypanosoma cruzi. Trans, roy. Soc. trop. Med. Hyg. 78.. 193-202(1984). Hoare C. A.: The trypanosomes of mammals. Blackwell Sci. Pubi.. Oxford/Edinburgh 1972. Hüther A. M.: Ultrastrukturelle Untersuchungen zum Einfluss von Cyclophosphamid auf experimentelle Trypanosoma crwrMnfektion. Tropenmed. Parasit. 32. 51-54 (1981). Kirch hoff L. V.. Engel J. C. Dvorak J. A.. Sher A.: Strains and clones ofTrypanosoma cruzi differ in their expression of a surface antigen identified by a monoclonal antibody. Molec. Biochem. Parasit. //. 81-89 (1984a). Kirchhoff L. V., Hieny S., Shiver M., Snary D.. Sher A.: Cryptic epitope explains the failure of monoclonal antibody to bind to certain isolates of Trypanosoma cruzi. J. immunol. Meth. 133. 2731-2735 (1984b). Marinkelle C. J.: The biology ofthe trypanosomes of bats. In: Biology ofthe Kinetoplastida. Vol. 1, ed. by W. H. R. Lumsden and D. A. Evans. Academic Press. London/New York/San Francisco 1976.. Marinkelle C. J.: Prevalence of Trypanosoma cruzi-likc infection of Colombian bats. Ann. trop. Med. Parasit. 76. 125-134 (1982). 12.

(10) Miles M. A., Toye P. J.. Oswalds. C. Godfrey D. G: The identification by isoenzyme patterns of two distinct strain-groups of 1'rypanosoma cruzi. circulating independently in a rural area of Brazil. Trans, roy. Soc. trop. Med. Hyg. 71. 217-225 (1977). Miles M. A., Povoa M. M.. Prata \.. Cedillos R. A., De Souza A. A.. Macedo V.: Do radically dissimilar Trypanosoma cruzi strains (Zymodemes) cause Venezuelan and Bra/than forms of Chagas' disease'.' Lancet /957/11. 1338-1340. Mühlpfordt H.: Vergleichende Kinetoplastmorphologie verschiedener Trypanosomaartcn unter besonderer Berücksichtigung von Trypanosoma cruzi. Tropenmed. Parasit. 26. 239-246 (1975). Mühlpfordt H.: Vergleichende elektronenmikroskopische Untersuchungen der Kinetoplastmorphologie von Fledcrmaustrypanosomen und Trypanosoma cruzi. Z. Parasitenk. 65. 95-101 (1981). Mühlpfordt H.. Ebert F.. Schottelius J.. Cedillos R.. Velasco J.. Mosca W.: Comparative characterization of Venezuelan Trypanosoma cruzi stocks by electron microscopy, isoelectrofocusing and lectin typing. Tropenmed. Parasit. 35. 11-14 (1984). Plata F.. Pons F. G.. Eisen H.: Antigenic polymorphism of Trypanosoma cruzi: clonal analvsis of trypomastigote surface antigens. Europ. J. Immunol. 14. 392-399 (1984). Schottelius J.: The identification by lectins of two strain groups of Trypanosoma cruzi. Z. Parasitenk. 68. 147-154 (1982). Schottelius J., Uhlenbruck G.: Comparative studies of Trypanosoma cruzi and Trypanosoma cruzilike stocks from different South American countries using lectins. Z. Parasitenk. 69. 727-736 (1983). Schottelius J.. Koch O.. Uhlenbruck G: Differentiation of Trypanosoma cruzi Chagas. 1909 and Trypanosoma vespertilionis Battaglia. 1904 by various lectins. Tropenmed. Parasit. 34. 89-92 (1983). Sher A.. Snary D.: Specific inhibition ofthe morphogenesis of Trypanosoma cruzi bv a monoclonal antibody. Nature (Lond.) 300. 639-640 (1982). Snary D., Ferguson M. A. J., Scott M. T., Allen A. K.: Cell surface antigens of Trvpanosoma cruzi: use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein. Molec. Biochem. Parasit. 3. 343-356 (1981). W.H.O.: Maladie de Chagas. Note d'information N1-' 20. 6-9 (1983).. 13.

(11)

(12)